A new SOD mimic, Mn(III) ortho N-butoxyethylpyridylporphyrincombines superb potency and lipophilicity with low toxicity

Zrinka Rajic1,a, Artak Tovmasyan1, Ivan Spasojevic2, Huaxin Sheng3, Miaomiao Lu3,4, AliceM. Li5, Edith B. Gralla5, David S. Warner3, Ludmil Benov6, and Ines Batinic-Haberle1,*

1Department of Radiation Oncology, Duke University Medical Center, NC 27710, USA2Department of Medicine, Duke University Medical Center, NC 27710, USA3Department of Anesthesiology, Duke University Medical Center, NC 27710, USA4Department of Anesthesiology, Second Affiliated Hospital, Zhengzhou University, Henan, China5Department of Chemistry and Biochemistry, UCLA, Los Angeles, CA 90095-1569, USA6Department of Biochemistry, Faculty of Medicine, Kuwait University, 13110 Safat, Kuwait

AbstractThe Mn porphyrins of kcat(O2˙−) as high as that of a superoxide dismutase enzyme, and ofoptimized lipophilicity have already been synthesized. Their exceptional in vivo potency is at leastin part due to their ability to mimic site and location of mitochondrial superoxide dismutase,MnSOD. MnTnHex-2-PyP5+ is the most studied among lipophilic Mn porphyrins. It is ofremarkable efficacy in animal models of oxidative stress injuries and particularly in centralnervous system diseases. However, when used at high single and multiple doses it becomes toxic.The toxicity of MnTnHex-2-PyP5+ has been in part attributed to its micellar properties, i.e. thepresence of polar cationic nitrogens and hydrophobic alkyl chains. The replacement of a CH2group by oxygen atom in each of the four alkyl chains was meant to disrupt the porphyrin micellarcharacter. When such modification occurs at the end of long alkyl chains, the oxygens becomeheavily solvated, which leads to a significant drop in the lipophilicity of porphyrin. However,when the oxygen atoms are buried deeper within the long heptyl chains, their excessive solvationis precluded and the lipophilicity preserved. The presence of oxygens and the high lipophilicitybestow the exceptional chemical and physical properties to Mn(III) meso-tetrakis(N-n-butoxyethylpyridinium-2-yl)porphyrin, MnTnBuOE-2-PyP5+. The high SOD-like activity is fullypreserved and somewhat enhanced: log kcat(O2˙−) = 7.83 vs 7.48 and 7.65 for MnTnHex-2-PyP5+

and MnTnHep-2-PyP5+, respectively. MnTnBuOE-2-PyP5+ was tested in an O2˙− - specific invivo assay – aerobic growth of SOD-deficient yeast, Saccharomyces cerevisiae, where it was fullyprotective in the range of 5 – 30 µM. MnTnHep-2-PyP5+ was already toxic at 5 µM, andMnTnHex-2-PyP5+ became toxic at 30 µM. In a mouse toxicity study, MnTnBuOE-2-PyP5+ wasseveral-fold less toxic than either MnTnHex-2-PyP5+ or MnTnHep-2-PyP5+.

© 2012 Elsevier Inc. All rights reserved.*Corresponding author. Ines Batinic-Haberle, Ph.D., Department of Radiation Oncology-Cancer Biology, Duke University MedicalCenter, Research Drive, 281b/285 MSRB I, Box 3455, Durham, NC 27710, Tel: 919-684-2101, Fax: 919-684-8718,ibatinic@duke.edu.aPresent address: Zrinka Rajic, Ph.D., Department of Medicinal Chemistry, Faculty of Pharmacy and Biochemistry, University ofZagreb, A. Kovacica 1, Zagreb, Croatia

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NIH Public AccessAuthor ManuscriptFree Radic Biol Med. Author manuscript; available in PMC 2013 May 01.

Published in final edited form as:Free Radic Biol Med. 2012 May 1; 52(9): 1828–1834. doi:10.1016/j.freeradbiomed.2012.02.006.

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IntroductionMn porphyrin-based SOD mimics, peroxynitrite scavengers and redox modulators ofcellular signaling pathways have been developed for over 20 years [1–5]. The optimizationof their structure took place in three phases. In Phase I, we aimed at achieving the potencyof SOD enzymes, i.e. the rate constant for the catalysis of O2˙− dismutation as close aspossible to log kcat(O2˙−) = 8.84–9.30. Over years we have explored a number of porphyrinsand established the structure-activity relationships (SAR) for cationic, neutral and anionicMn porphyrins [1–3]. The SAR relates the SOD-like potency (expressed in terms of kcat) tothe thermodynamic parameter, reduction potential for the MnIIIP/MnIIP redox couple, E1/2.In Figure 1 the SAR is shown for cationic MnPs only. The presence of charges on an SODmimic close to the Mn site in ortho positions of pyridyl rings affords both thermodynamicand kinetic facilitation for the O2˙− dismutation [1–3, 5].

Based on SAR and our simple O2˙−-specific in vivo model of (the aerobic growth of SOD-deficient E. coli), the ortho isomeric Mn(III) N-alkylpyridylporphyrins have emerged as themost potent and stable SOD mimics with log kcat ~ 8. Among them, MnTE-2-PyP5+ hasbeen the most frequently studied compound.

As research progressed it became obvious that in vivo efficacy depends upon thebioavailability of the compound also, i. e. its tissue, cellular and subcellular distribution,which in turn depends upon its lipophilicity, charge, size, shape, and bulkiness. In Phase II,while maintaining the total charge and its position, we lengthened the alkyl chains frommethyl to octyl. Such modification largely increased the efficacy of MnPs in amelioratingcentral nervous system injuries, such as stroke, subarachnoid hemorrhage, pain and cerebralpalsy [1, 6, 7]. Among the lipophilic analogs, MnTnHex-2-PyP5+ has been the mostfrequently studied porphyrin [1, 3, 6]. The remarkable efficacy of MnTE-2-PyP5+ andMnTnHex-2-PyP5+ in numerous animal models of oxidative stress diseases [1, 3, 8–10] maybe at least in part attributed to their ability to accumulate in mitochondria, mimickingtherefore the site and the action of critical mitochondrial enzyme, MnSOD [7].Mitochondrial accumulation of lipophilic MnTnHex-2-PyP5+ was significantly enhancedwhen compared to a more hydrophilic MnTE-2-PyP5+ [7].

In an ongoing Phase III we aimed at suppressing the toxicity of lipophilic N-alkylpyridylporphyrins. The toxicity of MnTnHex-2-PyP5+ at higher concentrations/doses isprimarily assigned to its micellar property, and thus ability to damage membranes [11, 12].We already observed that the replacement of a CH2 group by oxygen atom in each of thefour butyl chains suppressed the toxicity of MnTnBu-2-PyP5+, but unfortunately, it greatlydecreased its lipophilicity also [4]. We applied the same approach to the modification of alipophilic MnTnHex-2-PyP5+, attaching methoxy groups at the end of the hexyl chains,hoping that in this case the lipophilicity of the longer hexyl chains would outbalance thepolarity of oxygens. Yet, the methoxyhexyl derivative, MnTMOHex-2-PyP5+ issignificantly less lipophilic in comparison with its parent MnTnHex-2-PyP5+ [13]. In thiswork, we showed that if the oxygen atoms are buried within the long-alkyl chains (closer tothe pyridyl rings) of MnTnBuOE-2-PyP5+, and are thus protected from the extensivesolvation, the high lipophilicity is fully preserved. The toxicity of MnTnBuOE-2-PyP5+ toboth mice and Saccharomyces cerevisiae is greatly reduced relative to either MnTnHex-2-PyP5+ (bearing the same number of carbon atoms in pyridyl substituents) or MnTnHep-2-PyP5+ (of the same length of pyridyl substituents).

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ExperimentalGeneral

H2T-2-PyP was purchased from Frontier Scientific. 2-Butoxyethanol (>99%) was from TCIAmerica, p-toluenesulfonyl chloride (98%) from Alpha Aesar, and tetra-n-butylammoniumchloride hydrate (98%) was from Aldrich. MnCl2 × 4H2O (99.7%) was supplied by J. T.Baker, NH4PF6 (99.99% purity) was from GFS chemicals. Diethyl ether anhydrous andacetone were from EMD chemicals, while dichloromethane, chloroform, acetonitrile, EDTAand KNO3 were purchased from Mallinckrodt. N,N-dimethylformamide anhydrous (99.8%purity), pyridine anhydrous (99.8%) and plastic-backed silica gel TLC plates were fromSigma-Aldrich. Xanthine and equine ferricytochrome c (Lot 7752) were from Sigma,whereas xanthine oxidase was prepared by R. Wiley [5]. Sodium sulfate anhydrous (>99%)was from Sigma-Aldrich. All chemicals were used as received without further purification.The porphyrins (MnTE-2-PyP5+, MnTnHex-2-PyP5+, and MnTnHep-2-PyP5+) usedthroughout this study were synthesized according to the procedures described earlier [2].

Elemental analysesElemental analyses of the porphyrin ligand and its Mn complex were performed by AtlanticMicroLab (Norcross, GA, USA). All analyses were done in duplicates.

Uv/vis spectraUv/vis spectra were recorded in H2O at room temperature on UV-2501PC Shimadzuspectrophotometer with 0.5-nm resolution.

Electrospray ionization mass spectrometric (ESI-MS) analysesThe analyses were performed as described elsewhere [14] on Applied Biosystems MDSSciex 3200 Q Trap LC/MS/MS spectrometer at Duke Comprehensive Cancer Center, SharedResource PK Labs. All samples of 1 µM concentrations were prepared in acetonitrile:H2O(1:1, v/v; containing 0.01% v/v HFBA) mixture and infused for 1 min at 10 µl/min into thespectrometer (curtain gas 20 V, ion spray voltage 3500 V, ion source 30 V, t=300 °C,declustering potential 20 V, entrance potential 1 V, collision energy 5 V, gas N2) [15]. In thepresence of soft anion, heptafluorobutyric acid, which pairs with cationic porphyrin, nofragmentation of the porphyrin has been detected. Thus any partially quaternized porphyrinfound in mass spectrum is the consequence of partial alkylation or alkoxyalkylation, but notof fragmentation during spectral analysis [14].

Synthesis2-Butoxyethyl p-toluenesulfonate was prepared as already described [16]; spectroscopic datawere in agreement with the proposed structure.

H2TnBuOE-2-PyPCl4N-butoxyethylation was performed as previously described for analogous alkyl derivatives[17]. To a solution of H2T-2-PyP (40 mg, 0.0065 mmol) in anhydrous DMF (6.6 mL,preheated at 105 °C for 10 min) 2-butoxyethyl p-toluenesulfonate (8.8 g, 0.032 mol) wasadded. The course of the reaction was followed by TLC using 1:1:8 =KNO3(sat):H2O:CH3CN as a mobile phase. While atropisomers with lipophilic analogs ofthe ortho alkyl series separate on the TLC plate, under same conditions with H2TnBuOE-2-PyP4+ and its Mn complex they do not. Quaternization of sterically hindered ortho isomerwas completed in ~3.5 days, observed as a single spot on TLC plate. The reaction mixturewas filtered into the separatory funnel containing H2O and chloroform and extractedmultiple times with chloroform. After filtration of the combined H2O layers, the isolation of

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H2TnBuOE-2-PyP4+ as a chloride salt was performed as previously described [17].Elemental analysis: H2TnBuOE-2-PyPCl4 × 11.5 H2O. Anal Calcd for C64H101N8O15.5Cl4:C, 56.01; H, 7.42; N, 8.17; Cl, 10.33%. Found: C, 56.22; H, 7.02; N, 8.34; Cl, 10.21%.

MnTnBuOE-2-PyPCl5Metallation was achieved as previously described for alkyl analogs [17], with slightmodifications. The aqueous solution of H2TnBuOE-2-PyPCl4 was basified with 0.1 MNaOH to pH ~10.7, which ensured significant amount of reactive (OH)MnP species anddeprotonation of inner pyrrolic nitrogens, both essential for successful metallation at roomtemperature. Metallation was performed with 20-fold excess of MnCl2 × 4H2O upon whoseaddition the pH of the solution dropped to ~7.8 due to the extensive hydrolysis of Mn aquaion. The course of metallation was monitored by TLC using 1:1:8 = KNO3(sat):H2O:CH3CNas a mobile phase, as well as by uv/vis spectroscopy. The very useful means to follow thecompletion of the metallation is to follow the loss of the ligand fluorescence under UV lamp(~350 nm). Completion of the metallation was achieved within hours at room temperature.MnP solution was filtrated (to remove Mn oxo/hydroxo species and MnO2). The isolation ofMnTnBuOE-2-PyP4+ as a chloride salt was performed as previously described [17].Elemental analysis: MnTnBuOE-2-PyPCl5 × 12.5 H2O. Anal Calcd forMnC64H101N8O16.5Cl5: C, 51.98; H, 6.88; N, 7.58; Cl, 11.99%. Found: C, 52.07; H, 6.71;N, 7.83; Cl, 11.76%.

ElectrochemistryCyclic voltammetry was performed on CH Instruments model 600 voltammetric analyzerwith MnTE-2-PyP5+ as a standard as previously described in details [4, 18].

Catalysis of O2˙− dismutation (cytochrome c assay)The ability of MnTnBuOE-2-PyP5+ to dismute O2˙− was evaluated via cytochrome c assaywhich was proven to be equally effective as pulse radiolysis and stopped-flow methodology[2, 3, 19–22]. The experiments were conducted at room temperature (25 ± 1 °C) in 0.05 Mpotassium phosphate buffer, pH 7.8, 0.1 mM EDTA as reported elsewhere [19].

Stability of MnTnBuOE-2-PyP5+ towards HClWe aimed to see if the ether functionality would survive the stomach acidity when thecompound is given per os. MnTnBuOE-2-PyP5+ was tested at pH 1, at 4 h, 24h and 48 hafter acidification of ~ 0.5 mM and 5 µM solutions. Due to the high levels of chloride,molecular ions bearing mixed chloride/butyrate counteranions were found in mass spectrum.No changes were observed in uv/vis and ESI-MS spectra of MnP after acidification,indicating that neither loss of Mn nor the hydrolysis of ether bond happened.

LipophilicityLipophilicity was determined as retention factor on TLC plates, Rf and as log POW asdescribed earlier [15].

S. cerevisiae studyThe wild-type S. cerevisiae strain used was EG103, which has a complete genotype ofMATα, leu2, his3, trp1, ura3. The sod1Δ mutant S. cerevisiae strain, lacking Cu,ZnSOD,was EG118, which has a complete genotype of MATα, leu2, his3, trp1, ura3,sod1Δ::URA3. These strains, described by Gralla and Valentine [23], were first streakedfrom 20% glycerol stocks onto yeast extract, peptone agar supplemented with 2% dextrose(YPD) and incubated for 72 hr at 30 °C under hypoxic conditions [24]. Single colonies werethen selected to grow to stationary phase under hypoxic conditions at 30 °C in the above

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mentioned medium supplemented with penicillin and streptomycin to prevent bacterialgrowth. The overnight cultures were diluted ~1,000-fold with YPD medium. Absorbance at600 nm (A600) has been checked and adjusted to be equal for both parental and sod1Δmutant stock suspensions, and 200 µL aliquots were transferred into 96-well plates. Aqueoussolutions of Mn porphyrins were filter-sterilized (0.22-µm filter, Whatman, Middlesex, UK).Cultures in 96-well plates were grown aerobically at 30 °C and 250 rpm on a thermostaticshaker. All samples were run in triplicates and in controls the volume was compensated withsterile distilled water. Growth was followed turbidimetrically by measuring the absorbanceat 600 nm using ELISA reader.

Mouse toxicity study8–10 weeks old male C57BL/6J mice were used for the toxicity study. Mice body weightand rotarod performance were measured daily before and after MnP injections as describedearlier [25, 26]. Also, behavior and survival were closely observed immediately after thetreatment with MnPs. All animal procedures conformed to the Institutional Animal Care andUse Committee and National Institute of Health guidelines. Mice were given intraperitoneal(ip) injections with saline, 0.5, 1.5, 4.5 and 9 mg/kg/day of MnTnBuOE-2-PyP5+twice perday (b.i.d.) for 7 days. Mice were placed on a rolling bar at the accelerated speed of 4–40rpm and the latency to fall from the bar was automatically recorded. Data were expressed as% of the pre-treatment baseline. Four mice were used per dose. In order to more accuratelycompare the toxicity among MnTnBuOE-2-PyP5+, MnTnHex-2-PyP5+ and MnTnHep-2-PyP5+, additional four mice per dose were also injected with single injection of 2.5 and 5mg/kg MnTnHex-2-PyP5+, 5 and 10 mg/kg MnTnBuOE-2-PyP5+ and 2.5, 5 and 10 mg/kgMnTnHep-2-PyP5+.

Results and DiscussionWhile of remarkable efficacy at as low single or multiple doses as 0.05 mg/kg [1–3], alipophilic cationic MnTnHex-2-PyP5+ exerts toxicity at higher doses which is in part due toits micellar character. We hoped that the placement of oxygen atoms at the very end of thealkyl chains will not significantly diminish the porphyrin lipophilicity, but will disrupt themicellar character of the alkyl chains. Yet, this happened not to be the case: significant dropin the lipophilicity of MnTMOHex-2-PyP5+ (as measured via chromatographic retentionfactor, Rf) relative to MnTnHex-2-PyP5+ was observed (Figure 2). Moreover, duringmethoxyhexylation, the methoxyhexyl p-toluenesulfonate underwent intramolecularcyclization, which diminished the purity of the compound. Rather than interacting withpyridyl nitrogens only, the methoxyhexyl carbocation undergoes intramolecular cyclizationwith its own oxygen atom leading to the formation of a 7-membered ring. Subsequently, theoxygen-carbon bond of the methyl group weakens; in turn methyl group leaves andmethylates the pyridyl nitrogen. Due to the length of the quaternization process (~3.5 days),a significant amount of the species with 3 methoxyhexyl and one methyl groups were foundin the preparation. The mechanistic aspects of the intramolecular cyclization leading to theunwanted product will be detailed in a subsequent publication [Rajic et al, in preparation,27].

The quaternization of a meta isomer to form H2TMOHex-3-PyP4+ was much faster (withinhours). Subsequent metallation gave rise to MnTMOHex-3-PyP5+; the preparation containsup to 5% of the species bearing one methyl group. To understand the origin of much higheramount of the porphyrin with 3 methoxyhexyl groups and one methyl group, we synthesizeda number of porphyrins with different chain lengths and different positions of the oxygenatoms in the alkyl chains [Rajic et al, in preparation, 27]. The different position of theoxygens in alkoxyalkyl p-toluenesulfonate leads to the formation of cycles of different sizeand therefore different stability via intramolecular rearrangement; consequently more or less

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of the porphyrin species bearing one undesirable alkyl chain will be formed [27]. Suchstrategy led us to eventually explore the butoxyethyl p-toluenesulfonate, and we ended upwith beautiful molecule of superior properties: MnTnBuOE-2-PyP5+. With butoxyethyl p-toluenesulfonate, the intramolecular cyclization within +CH2-CH2-O-CH2-CH2-CH2-CH3group would be unfavorable due to the formation of unstable 3-membered ring.Consequently, insignificant amount of the porphyrin bearing 3 butoxyethyl groups and onebutyl group could be expected. Indeed, that was the case. Moreover, we were pleasantlysurprised that the position of oxygen atoms buried deeper into the alkyl chains partlysuppressed their solvation. Consequently, the molecule is similarly lipophilic asMnTnHex-2-PyP5+ and MnTnHep-2-PyP5+ (Figure 2). Moreover, as Mn ion approachesinner pyrrolic nitrogens, it makes transient bonds with oxygens, which greatly speeds up themetallation process. Transient bond formation, however, weakens oxygen-butyl bond, and inturn insignificant presence of the species with 3 butoxyethyl and one hydroxyethyl werefound in a final preparation. With porphyrins that contain long alkyl chains, the sterichindrance prevented the easy approach of Mn ion to the porphyrin ring; the metallation ofH2TnHex-2-PyP5+ occurs at 100 °C, while within few hours at room temperature in the caseof MnTnBuOE-2-PyP5+.

MnTnBuOE-2-PyP5+ and its metal-free ligand were characterized by thin-layerchromatography (Rf), elemental analysis, uv/vis spectroscopy, ESI-MS, and partitionbetween n-octanol and water (log POW). The uv/vis data, the molar absorption coefficientsand the corresponding λmax are given in Table 1, while the ESI-MS data are provided inTable 2.

The log kcat(O2˙−), and the E1/2 in mV vs NHE for MnIIIP/MnIIP redox couple are given inTable 3. As expected, the properties of MnTnBuOE-2-PyP5+ are dominated by the positivecharges located on the pyridyl nitrogens, and by the presence of oxygen atoms in pyridylsubstituents. When the pyridyl substituents are lipophilic, the cationic charges on nitrogensare not excessively solvated, and in turn exert stronger electron-withdrawing effect upon Mnsite: both MnTnHex-2-PyP5+ and MnTnHep-2-PyP5+ have E1/2 ≥ 100 mV positive thanMnTE-2-PyP5+, indicating the less electron-deficiency of the latter than of the former twoporphyrins (Table 3). However, due to the presence of oxygens in alkyl chains, butoxyethylchains are more solvated than hexyl or heptyl chains, which in turn hinders nitrogen charges(and thus suppresses their electron-withdrawing effect upon the Mn site; consequently, theE1/2 is less positive (Table 3). Additionally, the decrease in E1/2 may occur as a consequenceof the electron-donating properties of the alkoxyalkyl substituents. Further, the solvation ofthe porphyrin cavity formed by pyridyl substituents benefits the catalysis of O2˙−

dismutation as it involves the interaction of ionic species: singly charged Mn site andsuperoxide. Consequently, MnTnBuOE-2-PyP5+ is ~ 1.5–2-fold more able catalyst of O2˙−

dismutation than its alkyl analogs which either has the same number of carbon atoms inpyridyl substituents (MnTnHex-2-PyP5+), or whose pyridyl substituents are of the similarlength (MnTnHep-2-PyP5+) (Table 3).

The lipophilicity of Mn porphyrins has routinely and conveniently been measured by TLCretention factor, Rf. Rf is linearly related to the partition between the n-octanol and water,log POW for the ortho and meta Mn(III) N-alkylpyridylporphyrins [15]. Herein we aimed tosee if such relationship holds for the Mn(III) N-alkoxyalkylpyridylporphyrins also, and iftherefore Rf could be used as useful and convenient measure of their lipophilicity. Goodagreement was found between the lipophilicity described by either Rf or log POW for bothalkyl and alkoxyalkyl series of Mn porphyrins. Measured by log POW, and as expected dueto the presence of oxygen atoms, MnTnBuOE-2-PyP5+ is slightly less lipophilic thanMnTnHex-2-PyP5+ (Table 3).

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S. cerevisiae studyOur earlier studies indicated that E. coli is overly sensitive to the lipophilic Mn porphyrins.They exert toxicity at >1 µM concentrations, which is in part due to their high cellularaccumulation, micellar properties and different redox-activity of Mn site [11, 12, 31]. Thus,while efficacious at 1 µM, already at 5 µM MnTnHex-2-PyP5+ prevents E. coli growth. Wehave however observed that mammalian cells are less sensitive to lipophilic Mn porphyrins[32, 33]. Our preliminary data indicated that eukaryotic yeast is also much less sensitive tothe lipophilic Mn porphyrins and can tolerate well up to 30 µM MnTnHex-2-PyP5+ [34].The aerobic growth of SOD-deficient S. cerevisiae (lacking Cu,ZnSOD) is thus a simple andexcellent in vivo model of oxidative stress for the evaluation of the therapeutic potential oflipophilic Mn porphyrins. MnTnBuOE-2-PyP5+ fully supports S. cerevisiae growth at ≥5µM, and is of similar efficacy as MnTnHex-2-PyP5+; however, at 30 µM MnTnHex-2-PyP5+

becomes toxic (Figure 3). MnTnHep-2-PyP5+ is ~10-fold more lipophilic than MnTnHex-2-PyP5+ (Table 3). While of higher efficacy at only 0.5 and 1 µM than both MnTnHex-2-PyP5+ and MnTnBuOE-2-PyP5+, MnTnHep-2-PyP5+ becomes toxic at ≥ 5 µM.

Mouse toxicity studyIf injected ip at 2 mg/kg, MnTnHex-2-PyP5+ exerted transient toxicity; mice shivered andsat hunched for a while. The toxicity was more pronounced with 2.5 mg/kg, mice werebarely walking and sometimes showed tail twist. One of four mice died, but the othersrecovered to some extent on the day after injection. All 4 mice died on a day after single 5mg/kg ip injection of MnTnHex-2-PyP5+. However, all 4 mice injected with 5 mg/kg ofMnTnBuOE-2-PyP5+ were briefly quiet immediately after injection, but were running in thecage the day afterwards. Immediately upon the single ip injection of 10 mg/kg ofMnTnBuOE-2-PyP5+, the mice looked sleepy and did not like to walk; next day theyappeared better but not fully recovered. A tail twist and body shaking was seen with onemouse, but no mice died. Thus the toxicity of 10 mg/kg MnTnBuOE-2-PyP5+ appearssimilar or lower than that observed with 2.5 mg/kg of MnTnHex-2-PyP5+. As expected,MnTnHep-2-PyP5+ - with pyridyl substituents of equal length being a true analog ofMnTnBuOE-2-PyP5+ - was much more toxic than MnTnHex-2-PyP5+. All four mice whichreceived single ip injections of 10 mg/kg MnTnHep-2-PyP5+, 5 mg/kg MnTnHep-2-PyP5+,and 2.5 mg/kg MnTnHep-2-PyP5+, died within 10–15 minutes, 20–45 minutes and 24 hours,respectively. Taken together, the data (shown in Figure 4) indicate that MnTnBuOE-2-PyP5+ is at least 4 fold less toxic than MnTnHex-2-PyP5+ and MnTnHep-2-PyP5+.

MnTnBuOE-2-PyP5+ was then injected ip twice daily for the whole week at the doses up to4.5 mg/kg (9 mg/kg total dose per day); no signs of toxicity were observed as measured byrotarod and body weight loss (Figure 5). Even mice that underwent subarachnoidhemorrhage were fine with 2 daily injections of 4.5 mg/kg [Sheng et al, unpublished] - thesingle ip dose at which mice died if injected with hexyl or heptyl analogs. The total changein weight accounted for less than 5% when mice were injected with 9 mg/kg/day b.i.d. Withrotarod, improved performance was seen with almost all groups and at all times relative tothe 1st day of injections; however the changes did not reach statistical significance.

The lower toxicity of MnTnBuOE-2-PyP5+ relative to its hexyl and heptyl analogs is notfully understood at this point. The presence of oxygen atoms introduces the majordifferences among those compounds, and likely account to some extent for the substantialdifferences in their toxicity. Further experiments are under way to uncover possiblerelationships among the toxicity data, E1/2 values for MnIIIP/MnIIP redox couple, solvation,charge distribution and Mn-oxo chemistry of these water-soluble Mn porphyrin-basedtherapeutics.

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Due to the excellent properties of MnTnBuOE-2-PyP5+, we are intensifying efforts on itsclinical development. The assessment of its plasma, tissue, cellular and subcellular(mitochondrial and cytosolic) distribution is in progress, with the goal to understand thepossible impact of the presence of oxygen atoms on the bioavailability of MnTnBuOE-2-PyP5+ when compared to the MnPs of similar lipophilicity, MnTnHex-2-PyP5+ andMnTnHep-2-PyP5+.

ConclusionsWhile studying mechanistic aspects involved in the preparation of the alkoxyalkyl series ofMn porphyrins (MnTMOHex-2(and 3)-PyP5+), with the goal to reduce the toxicity oflipophilic N-alkylpyridylporphyrins, serendipity led us to the preparation of a new porphyrinmolecule with a high therapeutic potential, MnTnBuOE-2-PyP5+. The presence of theoxygen atoms and their positioning deeper within the hydrophobic long-alkyl chains makesthem less exposed to the solvent molecules, which in turn results in: (1) the preservedlipophilicity of the molecule; (2) the increased kcat due to the more solvated cavity aroundthe metal site; (3) the disrupted micellar character which in turn resulted in a greatlydiminished toxicity of MnTnBuOE-2-PyP5+ relative to its lipophilic alkyl analogs,MnTnHex-2-PyP5+ and MnTnHep-2-PyP5+ in both S. cerevisiae and mouse toxicity study.In a very simple O2˙− - specific in vivo model, MnTnBuOE-2-PyP5+ proved efficacious insupporting aerobic growth of SOD-deficient S. cerevisiae to the extent of a wild yeast straincounterpart.

Highlights

• Mn porphyrin-based SOD mimic, MnTnHex-2-PyP5+, was optimized to reduceits toxicity

• Introduction of oxygen atoms into all 4 hexyl chains gave rise to superb SODmimic MnTnBuOE-2-PyP5+

• Positioning of oxygen atoms deep within hexyl chains preserved lipophiliccharacter of MnTnHex-2-PyP5+

• MnTnBuOE-2-PyP5+ is twice more potent SOD mimic, but few fold less toxicthan MnTnHex-2-PyP5+

AcknowledgmentsAuthors acknowledge financial help from Duke University’s CTSA grant 1 UL 1 RR024128-01 from NCRR/NIH(AT, IBH, IS, ML, HS, DSW), W.H. Coulter Translational Partners Grant Program (IBH, IS, AT), and NIH/NCIDuke Comprehensive Cancer Center Core Grant (5-P30-CA14236-29) (IS) and IBH general research funds (ZR).IBH, IS and DSW are consultants with BioMimetix Pharmaceuticals, Inc. LB acknowledges the financial supportfrom Kuwait University, Grant MB01/09, and the excellent technical assistance of Milini Thomas.

Abbreviations

O2•− superoxide

SOD superoxide dismutase

H2TnBuOE-2-PyP5+ meso-tetrakis(N-(n-butoxyethylpyridinium-2-yl)porphyrin

MnPs Mn(III) N-alkyl- and N-alkoxyalkylpyridylporphyrins

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Mn(II)P, Mn(III)P Mn porphyrins with Mn in its +2 and +3 oxidation states

MnTE-2-PyP5+

(AEOL10113, FBC001)Mn(III) meso-tetrakis(N-ethylpyridinum-2-yl)porphyrin

MnTnBu-2-PyP5+ Mn(III) meso-tetrakis(N-n-butylpyridinum-2-yl)porphyrin

MnTMOE-2-PyP5+ Mn(III) meso-tetrakis(N-methoxyethylpyridinum-2-yl)porphyrin

MnTnHex-2-PyP5+ Mn(III) meso-tetrakis(N-n-hexylpyridinum-2-yl)porphyrin

MnTnHep-2-PyP5+ Mn(III) meso-tetrakis(N-n-hepylpyridinum-2-yl)porphyrin

MnTnOct-2-PyP5+ Mn(III) meso-tetrakis(N-n-octylpyridinum-2-yl)porphyrin

MnTMOHex-2-PyP5+ Mn(III) meso-tetrakis(N-(6’-methoxyhexyl)pyridinum-2-yl)porphyrin

MnTMOHex-3-PyP5+ Mn(III) meso-tetrakis(N-(6’-methoxyhexyl)pyridinum-3-yl)porphyrin

MnTnBuOE-2-PyP5+

(BMX-001)Mn(III) meso-tetrakis(N-n-butoxyethylpyridinium-2-yl)porphyrin

MnTnBuOE-3-PyP5+ Mn(III) meso-tetrakis(N-n-butoxyethylpyridinium-3-yl)porphyrin

Rf thin-layer chromatographic retention factor that presentsthe ratio between the solvent and compound path in 1:1:8= KNO3(sat):H2O:CH3CN solvent system

E1/2 half-wave reduction potential

SOD superoxide dismutase

NHE normal hydrogen electrode

log POW partition between n-octanol and water

ip intraperitoneal

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Figure 1.Structure-activity relationship between the redox property of cationic Mn porphyrins, E1/2(reduction potential for the MnIIIP/MnIIP redox couple) and SOD-like potency, logkcat(O2˙−). The data for the SOD enzyme are also shown. The optimized SOD mimics withregards to metal/ligand stability and log kcat are ortho Mn(III) N-substitutedpyridylporphyrins with E1/2 in between +200 mV and +400 mV vs NHE.

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Figure 2.The lipophilicity of Mn(III) N-alkyl- and N-alkoxyalkylpyridylporphyrins as determined byTLC chromatographic retention factor. The presence of the oxygen atoms at the periphery ofthe alkyl chains in MnTMOHex-2-PyP5+ results in their solvation and in turn causes largedrop in the lipophilicity relative to MnTnHex(nHep)-2-PyP5+. However, when the pyridylsubstituents in MnTnBuOE-2-PyP5+ are equally long but contain oxygens closer to theporphyrin ring, the solvation is minimized and therefore the lipophilicity not significantlyaffected relative to MnTnHex(nHep)-2-PyP5+.

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Figure 3.The growth of the wild type (EG 103) and SOD-deficient (sod1Δ) S. cerevisiae (EG118) inthe presence and absence of lipophilic Mn porphyrins. The most efficacious is the mostlipophilic compound, MnTnHep-2-PyP5+ (Table 3), presumably due to its highestaccumulation in the cell, but is therefore also more toxic at ≥5 µM. MnTnHex-2-PyP5+

which is routinely toxic to SOD-deficient E. coli at >1 µM [11, 31] is protective to SOD-deficient S. cerevisiae at up to 10 µM, and becomes toxic at 30 µM. MnTnBuOE-2-PyP5+

fully supports S. cerevisiae growth in the range of 5 to 30 µM.

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Figure 4.Comparison of Mn(III) N-butoxyethylpyridylporphyrin to its alkyl analogs with respect tothe mice toxicity when given via single ip injection.

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Figure 5.Five groups of mice were treated with saline, 0.5, 1.5, 4.5 and 9 mg/kg/day ip (given in twoincrement, b.i.d.) for a week. Mice performance on rotarod and their weight were followedover a week. No significant toxicity of drug as measured by these two parameters.

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Table 1

Uv/vis absorption maxima, λmax (nm) and molar absorption coefficients (ε, M−1 cm−1) of H2TnBuOE-2-PyPCl4 and its Mn(III) complex.

(Metallo)porphyrin λmax nm (log ε)a

H2TnBuOE-2-PyPCl4 263.50 (4.39), 418.00 (5.32), 513.50 (4.23), 544.50 (3.63), 586.00 (3.83), 638.50 (3.40)

MnTnBuOE-2-PyPCl5 213.50 (4.75), 262.50 (4.60), 365.00 (4.75), 412.00 (4.42), 455.00 (5.25), 559.50 (4.17), 783.00 (3.33)

aSpectra were taken in water at room temperature. Molar absorption coefficients (M−1 cm−1) were determined within 5 % errors. λmax (nm)

were determined with errors inside ± 0.5 nm.

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Table 2

Electrospray mass spectrometry (ESI-MS) data for H2TnBuOE-2-PyP4+ (H2P4+) and its Mn(III) complex

(MnP5+).

Species m/z

Found Calcd

H2P4+/4 256.1 255.7

(H2P4+− H+)3+/3 340.9 340.5

(H2P4+ + HFBA−)3+/3 412.2 411.9

(H2P4+ + HFBA− − H+)2+/2 617.7 617.3

(H2P4+ + 2HFBA−)2+/2 724.7 724.3

(H2P4+ + 2HFBA− + H2O)2+/2 732.6 733.3

(H2P4+ + 3HFBA− + H+)2+/2 831.6 831.3

(MnP5+ + HFBA−)4+/4 322.3 322.1

(MnP5+ + 2HFBA−)3+/3 501.0 500.5

(MnP5+ + 2HFBA− + H2O)3+/3 506.2 506.5

(MnP5+ + 3HFBA−)2+/2 857.6 857.2

(MnP5+ + 3HFBA− + H2O)2+/2 865.7 866.2

a~1 µM solution of porphyrin and metalloporphyrin in 1 : 1 v/v acetonitrile : H2O (containing 0.01% v/v heptafluorobutyric acid (HFBA)) mixture,

20 V cone voltage.

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Table 3

Metal-centered reduction potential E1/2 vs NHE (for MnIIIP/MnIIP redox couple), log kcat for O2˙−

dismutation of Mn porphyrins, and the lipophilicity of Mn porphyrin-based SOD mimics expressed in terms ofpartition between n-octanol and water, log POW.

Mn porphyrin E1/2/mV vs NHE a log kcatb Rf log POW

d

MnTE-2-PyP5+ +228 7.76 (cyt c) 7.73 (p.r.)c 0.055 −7.79

MnTnBu-2-PyP5+ +254 7.25 0.209 −6.19

MnTnHex-2-PyP5+ +314 7.48 0.355 −3.84

MnTnHep-2-PyP5+ +342 7.65 0.427 −3.18

MnTnOct-2-PyP5+ +367 7.71 0.500 −2.32

MnTMOE-2-PyP5+ +251 8.04 (p.r.)c 0.082 −7.52

MnTMOHex-3-PyP5+ +68 6.78 0.390 −5.45

MnTnBuOE-2-PyP5+ +277 7.83 0.423 −4.10

SOD enzymese ~ +300 8.84–9.30

aE1/2 is determined in 0.05 M phosphate buffer (pH 7.8, 0.1 M NaCl);

bkcat was determined by cytochrome c assay in 0.05 M potassium phosphate buffer (pH 7.8, at 25 ± 1 °C);

cp.r., pulse radiolysis;

ddata obtained from the relationship Rf vs log POW (log POW = 12.207 × Rf – 8.521), and direct determinations of log POW for Mn(III) N-

alkoxyalkylpyridylporphyrins [7];

ethe data are taken from the references [28–30].

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